Preparation of Stabilized Catalase Enzymes Using Polyvinyl Alcohol

ABSTRACT

There is provided a method of producing a stabilized catalase enzyme. In the method, a substrate is thoroughly mixed with phosphate borate and catalase, rinsed with water and the solids dried. The dried solid may be mixed with polyvinyl alcohol and dried for further stabilization. The stabilized powder may be mixed with various skin solutions (lotions, ointments and the like). The catalase enzyme can catalyze the reaction of peroxide to oxygen.

This application claims priority from U.S. provisional patentapplications 61/664,251 filed on Jun. 26, 2012 and from 61/769,395 filedFeb. 26, 2013, respectively.

BACKGROUND

The present disclosure relates to a method of stabilizing catalaseenzymes for longer term storage and stability until use. This disclosurealso relates to the stabilized enzymes.

Oxygen is essential to sustaining life. Marine life utilize oxygen indissolved form whereas land based species including humans utilizegaseous oxygen. The lack of oxygen or hypoxia is commonly experienced bypeople in their extremities (e.g. feet) as they get older due to poorblood circulation as well as by those with conditions such as diabetes.Studies have also shown below normal, low oxygen tension in the skins ofolder people. This often leads to poor skin health and an excessivepresence of visible conditions such as wrinkles, dryness and lower skinelasticity. Over the years, cosmetic manufacturers have introduced skinformulations with a large variety of ingredients such as emollients,exfoliators, moisturizers etc., to retard these age related effects andimprove and maintain skin health. Few formulations have focused on thedirect delivery of oxygen to the skin.

Oxygen delivery to the skin has been examined for medical use, e.g. intreating of the compromised skin (wounds, inflammation and trauma) andmore recently, intact skin. For example, Ladizinsky patented an oxygengenerating wound dressing (U.S. Pat. No. 5,792,090). More recently,Gibbins et al. patented a method of making an oxygen generating foamdressing based on a polyacrylate polymer (U.S. Pat. No. 7,160,553).While the method of making an oxygen generating foam dressing isstraightforward, the dressing itself suffers from a few drawbacks. Forinstance, the shelf life of the dressing is insufficient because oxygenfrom the dressing diffuses out of the foam cells over time. Analternative to the foam dressing in the form of an on-demand oxygengenerating topical composition was proposed to overcome the limitationof the short shelf life (Ladizinsky US2009/0074880). In the '880publication, a gel containing a catalyst and a peroxide in a separatereservoir, are brought together immediately before applying the mixtureto the skin and covering it to maintain contact with the skin. Whetherused for cosmetic applications or medical applications, oxygengeneration is generally achieved though the catalytic decomposition of aperoxide, commonly hydrogen peroxide.

In any of the applications using catalyst and peroxide, a problem thathas been found is that the catalyst can become inactivated duringstorage in a short period of time. Elevated temperatures accelerate thisinactivation for many catalysts. For modern shipping and customer usage,it is important that the product be stable for a period of timesufficient to package, ship, market and sell it and to be stable in theuser's home or other location. The stabilization of peroxide and/or acatalyst in a composition would be a step forward that would allow longterm storage of the product. It would also be desirable if the productwere stable at elevated temperatures commonly found in the shippingindustry.

There is a need for a way of stabilizing a catalyst and/or peroxide forextended periods of time and at elevated temperatures. This would allowfor the production, packaging, storage and shipping of a product withoutthe product becoming deactivated before the customer was able to use it.

SUMMARY

There is provided a way of stabilizing a catalyst, particularlycatalase, so that it may remain stable for an extended period of time.There is also provided a way of stabilizing a catalyst at elevatedtemperatures.

In the method, a substrate such as cellulose is thoroughly mixed withphosphate borate and catalase, rinsed with water and the solids dried.The dried solid may be mixed with polyvinyl alcohol and dried forfurther stabilization. The stabilized powder may be mixed with variousskin solutions (lotions, ointments and the like). The catalase enzymecan catalyze the reaction of peroxide to oxygen.

DETAILED DESCRIPTION

Described below are methods of stabilizing catalase so that it may bestored without becoming deactivated. Catalase, an enzyme commonlyproduced by bacteria and fungi, can be used as a catalyst to decomposeperoxide to oxygen. This decomposition is extremely rapid, but doesdepend on having a sufficient amount of catalase for a given amount ofperoxide in order to be successful. Catalase can easily becomeinactivated over time so stabilizing the catalase can extent its usefullifetime and improve its commercial viability. Stabilization at highertemperatures is also important since temperatures experienced duringshipping can be high enough to inactivate many catalysts.

The following procedure is a commonly accepted method of measuringcatalase activity that is used to determine how well the catalasemaintains its activity after stabilization and storage. After that areexamples of the preparation of the disclosed stabilized catalase. Notethat although the examples use microcrystalline cellulose as thesubstrate, any suitable substrate may be used, including ceramics andmetals.

Analyzing for Catalase Activity

The activity of catalase enzyme is defined in International Units (IU).A solution or solid powder (in suspension) is defined to have anactivity of one IU/ml or g if it can decompose 1 micromole of hydrogenperoxide per ml per minute at 25 C and pH 7. During the analysis forcatalase activity, the hydrogen peroxide concentration is preferablymaintained between 10 and 50 mM.

The analytical procedure for measuring catalase activity isstraightforward and is known to those of ordinary skill in the enzymeindustry. Briefly, following the addition of catalase solution ofunknown concentration to the hydrogen peroxide solution, the peroxideabsorbance value at 240 nm is monitored over time using a UV visiblespectrophotometer. Since the optical density is linearly related toperoxide concentration, using the absorbance versus time data, theconcentration of peroxide versus time data is obtained. Note that themolar extinction coefficient of hydrogen peroxide at 240 nm is 39.4liter/mol-cm. From the kinetic data, the initial rate (at time 0) isobtained and used to calculate the catalase activity.

Example 1 (Comparative) Preparation of Cellulose Coated with Catalasewith Poly Vinyl Alcohol (PVA) (CCP) Over-Coat (Non-Adsorption Method)

We report on a bench scale preparation method for cellulose catalasecomposite over-coated with PVA (hereafter referred to as CCP) that didnot involve a prolonged adsorption step. The rationale was to learn ifone could make robust CCP in a rapid manner; something that iscommercially always desirable.

Briefly, microcrystalline cellulose powder (6.0 g, Avicel® PC 105 fromFMC Biopolymer) was placed in a petri-dish. To the cellulose, asufficient amount of catalase solution (Grade 1500L, Activity: 50,000IU/ml from BIO-CAT Inc. of Troy, Va.) was added for a target theoreticalactivity of CCP of ˜10,000 IU/g. After swirling the slurry in thepetri-dish for 15 minutes, the dish was placed in a vacuum chamber toremove moisture and dry the powder. Periodically, the weight of thepetri-dish was checked. When no change in its weight was observed, thevacuum was discontinued. The dish was re-weighed and yielded ˜5.98 g ofCCP powder. The activity of CCP was found to be 2922 IU/g (See thegeneral description of catalase activity measurement).

In the next step, sufficient quantity of 2.4% w/w PVA solution (PVA 98+%hydrolyzed, MW: 85K-124K from Sigma Aldrich) was added to the CCPcorresponding to a PVA/cellulose mass ratio of 0.02. Once again, theslurry in the petri-dish was swirled for 15 minutes. Thereafter, thedish was returned to the vacuum chamber for removal of the solvent. Whenno change in the dish weight was observed (it took several hours), thevacuum was discontinued.

The dry CCP was scraped off the dish surface and transferred to a vialand stored at 4 C. The activity of CCP was measured to be 527 IU/g.Thus, following PVA coating and drying there was significant loss ofactivity of CCP.

To understand the aging effect, dry samples of CCP powder weremaintained at 4 C, 25 C and 40 C for 1 week and then their activitieswere re-measured. At 25 C and 40 C, the values were 344 IU/g and 99 IU/grespectively registering 35% and 81% loss. At 4 C, the measured value of441 IU/g indicated a loss of 16%. From the activity results, it isobvious that simply preparing CCP by simply blending respectiveingredients and then drying the resulting mix did not yield a robust CCPprototype.

Example 2 Preparation of Cellulose Powder Adsorbed with CatalaseOver-Coated with PVA

Rather than merely mixing catalase, cellulose powder and PVA, here wedescribe a method of preparing microcrystalline cellulose powderadsorbed with catalase enzyme and then over-coated with a thin coatingof PVA for protection (CCP-A). The method is identical to making CCP asdisclosed in Example 1 with the exception of how the catalase is appliedto the cellulose powder.

In an empty pre-weighed conical bottom polypropylene (PP) tube (from BDFalcon), a weighed quantity (0.5 g) of microcrystalline powder (Avicel®PC105) was added. This addition was followed by 4.5 ml phosphate boratebuffer (0.05M, pH: 6.7) and 0.5 ml diluted catalase solution having anactivity of 5000 U/ml. The diluted solution was prepared from a catalasestock solution (Grade 1500 L grade) having an activity of ˜50,000 U/ml.The contents were briefly mixed on a vortex mixer and the tube wasplaced on a shaker set at 800 rpm for 24 hours.

After 24 h, the liquid from the tube was drained and the solids wererinsed three times using 5 ml de-ionized water each time. After eachrinse, the liquid was discarded. After the third rinse, 1 ml of 1% w/wPVA solution was added to the wet cellulose solids in the tube and thecontents were mixed uniformly on a vortex mixer. The resultingsuspension was poured into a petri-dish. Any remaining cellulose in thetube was re-suspended by adding 2 ml de-ionized water and the suspensionwas transferred to the petri-dish.

The liquid in the petri-dish was allowed to air dry overnight at roomtemperature overnight inside a ventilated hood. The dry CCP-A powder wasgently scraped of the dish surface with a blunt knife and weighed (0.4g). The yield of CCP-A on cellulose weight basis was ˜80%.

In this example a number of catalase activity measurements were carriedout. First, the catalase activity for wet cellulose was measured (2282U/g). Second, after drying, the activity of resulting CCP-A was measuredat 1980 U/g. This is about a 13% loss in activity upon drying but thisstill was considered reasonable when compared with the results seen inExample 1.

Example 3 Preparation of Skin Hydrator Blend with CCP-A @ 500 IU/gActivity

The objective of this test was to (i) prepare CCP-A sample, (ii) blendthe CCP-A into Skin Hydrator lotion and (iii) subject the Skin Hydratorblend with CCP-A to thermal cycling (to simulate shipping transit)followed by accelerated age testing corresponding to a 2 years shelflife. The Skin Hydrator used herein is formula 1553-07 from BenchmarkLaboratories of Fountain Valley, Calif., though it is believed that thisprocedure can be used with virtually any skin lotion, ointment or thelike.

Preparation of CCP-A Powder

The following ingredients were added to a 15 ml PP conical tube andplaced on a shaker at room temperature to effect adsorption of catalaseon cellulose.

Microcrystalline cellulose 0.55 g BIO-CAT 1500 L 1.67 ml Phosphatebuffer 3.33 ml

After the adsorption step, a procedure identical to that in Example 2was followed to obtain CCP-A powder. In all, four batches were made andafter pooling the batches yielded roughly 2.5 g of powder. Based oncellulose, the yield was >100% but this was the result of moisture,about 10%, that was present even after prolonged drying. Because thepowder obtained was free flowing, it was used without further processingin the next step. The catalase activity of the pooled sample of CCP-Awas measured at 5103 IU/g.

Preparation of Skin Hydrator Blend with CCP-A

Dry CCP-A powder (1.5 g) was blended into Skin Hydrator lotion (13.5 g)at 10% w/w loading to obtain starting catalase activity of ˜500 IU/g inthe sample. Because of the difficulty of measuring catalase activity inthe lotion, the presence of catalase was confirmed indirectly byquantifying the decomposition of hydrogen peroxide after mixing it withO2 Reservoir lotion 1574-06 from Benchmark in 1:1 ratio. Freshly madeSkin Hydrator lotion with catalase decomposed 100% peroxide in slightlymore than 5 minutes. (The pass criterion was a minimum of 60%decomposition after 20 minutes.)

Thermal Cycling and Accelerated Aging of Skin Hydrator Lotion with CCP-Aat 500 IU/g Activity

Abbreviated thermal cycling was used because the cold condition (−20 C)was of no consequence as catalase is known to degrade at temperaturesabove 37 C. In the abbreviated thermal cycling, the lotion sample wasaged at 40 C for 72 h followed by 55 C for 6 h. Next, the sample wasplaced in an oven set to 35 C and monitored for its efficacy todecompose peroxide over 16 weeks for accelerated aging. As a cosmeticindustry norm, aging a product at 35 C for 16 weeks is considered areasonable estimate of 2 years of real time shelf life. A second samplethat served as control was maintained at room temperature (˜25 C).

The table below shows the percentage peroxide decomposition values over16 weeks. Note not all values are >60% which was set as the passcriterion. Nonetheless they are close to 60% within experimental error.Thus, we can conclude that Skin Hydrator lotion sample with CCP-A hasdemonstrated ability to withstand shipping transit and has met the twoyear shelf life criterion.

TABLE 1 Accelerated Aging Test Results of Skin Hydrator Lotion SamplesCCP- A @ 500 IU/g After Thermal Cycling @40 C/72h and 55 C/6h Sample WkWk Wk Wk Wk Wk Wk Wk Wk ID Temp Start Wk 1 Wk 2 Wk 3 4 5 6 7 8 9 10 1112 0191- RT 100% 69% 67% 65% 66% 66% ND 62% ND 60% ND 65% ND 66/500 IU35C 100% 69% 65% 63% 62% 63% ND 56% ND 55% ND 59% ND Sample ID Temp Wk13Wk14 Wk15 Wk16 0191- RT 70% ND ND 65% 66/500 IU 35C 68% ND ND 64% ND-Notdetermined

Example 4 Preparation of Skin Hydrator Blend with CCP-A @ 1000 IU/gActivity

CCP-A was prepared in a manner similar to the Example 3 except for thefollowing changes.

Microcrystalline cellulose 2.00 g BIO-CAT 1500 L 2.00 ml Phosphatebuffer 3.00 mlThe resulting CCP-A powder had an enzymatic activity of 21,000 IU/g.

A blend of Skin Hydrator with adsorbed catalase was prepared by blendingcellulose powder (1.0 g) into the base Skin Hydrator lotion (19.0 g)giving a starting catalase activity of ˜1000 IU/g, twice the value forthe samples in Example 3. The sample was subjected to thermal cycling asin Example 3 and then thermally aged at 35 C with an identical controlsample maintained at 25 C. As before, each week, the sample efficacy todecompose peroxide was monitored for a period of 16 weeks. The resultsobtained as percentage decomposition values are listed in the tablebelow.

TABLE 2 Accelerated Aging Test Results of Skin Hydrator Lotion SamplesWith CCP-A @ 1000 IU/g After Thermal Cycling @40 C/72h and 55 C/6hSample ID Temp Start Wk 1 Wk 2 Wk 3 Wk 4 Wk 5 Wk 6 Wk 7 Wk 8 Wk 9 Wk 10Wk 11 Wk 12 0191- RT 100% 90% 85% 81% 86% 85% ND 87% ND 83% ND 85% ND68/100 0 IU 35C 100% 90% 80% 79% 76% 76% ND 77% ND 80% ND 80% ND SampleID Temp Wk13 Wk14 Wk15 Wk16 0191- RT 90% ND ND 91% 68/100 0 IU 35C 85%ND ND 91% ND-Not determined

The results of peroxide decomposition for the entire duration areconsistently above 60% thus handily passing the set criterion. Suchoutstanding stabilization effect on catalase in a very hostileenvironment has never been demonstrated before to our knowledge.Comparing the percentage decomposition results in Examples 3 and 4, itseems one would choose to have the starting catalase activity in arobust commercial Skin Hydrator lotion to be somewhere between 500 and1000 IU/g for a 2 year shelf life. It is possible that the lotion samplein this Example could have exhibited the same efficacy for peroxidedecomposition beyond 16 weeks, though it was not tested. With such longshelf life the present lotion prototype has already outperformed anyknown catalase containing product currently on the market.

As illustrated by Examples 2-4 above, there is herein provided a methodof preparing stabilized microcrystalline cellulose through the steps ofthoroughly mixing microcrystalline cellulose powder, phosphate borateand catalase enzyme to create a mixture having solids and liquid. Theliquid is then drained from the mixture and the remaining solids arerinsed with water. Generally speaking, the ratio of cellulose to borateor catalase enzyme is between 1 and 10 and the ratio of borate tocatalase enzyme is between 0.5 and 10. The resulting enzyme has anactivity at 25 C between 500 IU/g and 1000,000 IU/g.

The examples also show that a dry, flowable powder containing catalasemay be made by the methods herein. Catalase is known to be difficult tostabilize in a dry form so this straight forward method provides anadvancement to the art of catalyst stabilization.

In addition to the stabilization of catalase as described herein, it hasalso been unexpectedly found that among the catalase derived fromdifferent organisms, the one derived from a fungus, Aspergillus niger,was most stable to thermal and chemical environments encountered. Thecatalase in buffered solution, in the adsorbed state on celluloseresisted degradation by heat or chemicals ingredients in cosmeticcompositions and retained the necessary activity to produce compositionsthat survived rigorous shipping protocols and prolonged ageing underheat to simulate accelerated ageing.

Those ordinarily skilled in the art will recognize numerous strains offungus are commercially available, though a desirable catalase source isfungus Aspergillus niger. It is important to note that this disclosureencompasses catalase derived from any source, including any fungalstrain. The catalase molecules derived from A. niger, however, have beenknown to contain manganese atoms. Catalase may also be derived fromgenetically modified organisms where the catalase producing vector maybe derived from A. niger or fungus in general and the host organisms inwhich the vector is inserted may be a fungus or another organism. Thus,in a broader aspect, the present disclosure encompasses catalase havingmanganese atom or atoms within its molecular structure regardless ofwhich fungal or other organisms it is derived from. Catalase withmanganese atoms in its molecular structure and having molecular weights<500,000 daltons are desirable.

1-5. (canceled)
 6. A microcrystalline cellulose adsorbed with a catalase enzyme and further comprising polyvinyl alcohol, wherein the catalase enzyme adsorbed on the microcrystalline cellulose with the polyvinyl alcohol exhibits a decreased loss in catalase activity compared to a catalase enzyme not adsorbed on the substrate with the polyvinyl alcohol, wherein the catalase enzyme adsorbed on the microcrystalline cellulose with the polyvinyl alcohol has an activity at 25° C. between 500 IU/g, and 1,000,000 IU/g, and wherein the catalase enzyme adsorbed on the microcrystalline cellulose with the polyvinyl alcohol is stable for up to 16 weeks at 35° C. after thermal cycling at 40° C. for 72 hours and 55° C. for 6 hours.
 7. A skin hydrator lotion comprising lotion and the microcrystalline cellulose of claim
 6. 8. (canceled)
 9. The microcrystalline cellulose of claim 6, wherein the ratio of the microcrystalline cellulose to the catalase enzyme adsorbed on the microcrystalline cellulose is between 1 and
 10. 10. The microcrystalline cellulose of claim 6, wherein the catalase enzyme adsorbed on the microcrystalline cellulose with the polyvinyl alcohol includes manganese atoms.
 11. The microcrystalline cellulose of claim 6, wherein the catalase enzyme adsorbed on the microcrystalline cellulose with the polyvinyl alcohol has a molecular weight of less than 500,000 Daltons.
 12. The microcrystalline cellulose of claim 6, wherein the catalase enzyme adsorbed on the microcrystalline cellulose with the polyvinyl alcohol is derived from fungus.
 13. The microcrystalline cellulose of claim 12, wherein the fungus is Aspergillus niger.
 14. The microcrystalline cellulose of claim 6, wherein the polyvinyl alcohol has a molecular weight between 85,000 and 124,000 Daltons.
 15. The microcrystalline cellulose of claim 6, wherein the catalase enzyme adsorbed on the microcrystalline cellulose with the polyvinyl alcohol can decompose from above 60% to 91% hydrogen peroxide after thermal cycling when the ratio of the catalase enzyme to hydrogen peroxide is 1:1.
 16. The microcrystalline cellulose of claim 6, wherein the microcrystalline cellulose further comprises phosphate and borate. 